Genes and proteins for aromatic polyketide synthesis

ABSTRACT

Nucleic acid molecules encoding polypeptides having polyketide synthase activity have been identified and characterized. Expression or over-expression of the nucleic acids alters levels of cannabinoid compounds in organisms. The polypeptides may be used in vivo or in vitro to produce cannabinoid compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/438,291, filed Feb. 21, 2017, which is a continuation of U.S. patentapplication Ser. No. 13/641,272, filed Oct. 15, 2012, which is aNational Stage Entry of International Application No. PCT/CA2011/000428,filed Apr. 15, 2011, which claims the benefit of U.S. Provisional PatentApplication USSN 61/324,343, filed Apr. 15, 2010, all of which areincorporated herein by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“P46230US02_SequenceListing.txt” (2,719 bytes), created on Jul. 31,2018, is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to nucleic acid molecules and proteinsinvolved in the synthesis of aromatic polyketides, and to uses of thenucleic acid molecules and proteins for producing cannabinoid compounds,and analogs thereof, and for altering cannabinoid production inorganisms.

BACKGROUND OF THE INVENTION

Cannabis sativa L. (cannabis, hemp, marijuana) is one of the oldest andmost versatile domesticated plants, which today finds use as source ofmedicinal, food, cosmetic and industrial products. It is also well knownfor its use as an illicit drug owing to its content of psychoactivecannabinoids (e.g. Δ⁹-tetrahydrocannabinol, Δ⁹-THC). Cannabinoids andother drugs that act through mammalian cannabinoid receptors are beingexplored for the treatment of diverse conditions such as chronic pain,multiple sclerosis and epilepsy.

Cannabinoids have their biosynthetic origins in both polyketide andterpenoid metabolism and are termed terpenophenolics or prenylatedpolyketides (Page J., Nagel J. (2006) Biosynthesis of terpenophenolicsin hop and cannabis. In J T Romeo, ed, Integrative Plant Biochemistry,Vol. 40. Elsevier, Oxford, pp 179-210.). Cannabinoid biosynthesis occursprimarily in glandular trichomes that cover female flowers at a highdensity. Cannabinoids are formed by a three-step biosynthetic process:polyketide formation, aromatic prenylation and cyclization (see FIG. 1).

The first enzymatic step in cannabinoid biosynthesis is the formation ofolivetolic acid by a putative polyketide synthase enzyme that catalyzesthe condensation of hexanoyl coenzyme A (CoA) and malonyl CoA. A TypeIII polyketide synthase, termed “olivetol synthase” and referred toherein as polyketide synthase/olivetol synthase (CsPKS/olivetolsynthase), from Cannabis sativa has recently been shown to form olivetoland several pyrone products but not olivetolic acid (Taura F, Tanaka S,Taguchi C, Fukamizu T, Tanaka H, Shoyama Y, Morimoto, S. (2009)Characterization of olivetol synthase, Type III a polyketide synthaseputatively involved in cannabinoid biosynthetic pathway. FEBS Lett. 583:2061-2066.). The nucleotide sequence of the gene encoding CsPKS/olivetolsynthase is found in GenBank under accession number AB164375 with thepolypeptide as accession BAG14339. The aforementioned products includethe pyrones hexanoytriacetic lactone (HTAL) and pentyldiacetic lactone(PDAL). The reason for the inability of this enzyme to form olivetolicacid, which is clearly a pathway intermediate based on the carboxylatestructure of the cannabinoids, is not known. The lack of olivetolic acidformation by this polyketide synthase from cannabis was confirmed by theinventors, as further described herein and also by Marks et al. (Marks MD, Tian L, Wenger J P, Omburo S N, Soto-Fuentes W, He J, Gang D R,Weiblen G D, Dixon R A. (2009) Identification of candidate genesaffecting Delta9-tetrahydrocannabinol biosynthesis in Cannabis sativa. JExp Bot. 60, 3715-3726.).

The second enzymatic step is the prenylation of olivetolic acid to formcannabigerolic acid (CBGA) by the enzymegeranylpyrophosphate:olivetolate geranyltransferase. This enzyme is anaromatic prenyltransferase and is the subject of commonly owned U.S.Provisional patent applications U.S. Ser. No. 61/272,057 filed Aug. 12,2009 and U.S. Ser. No. 61/272,117 filed Aug. 18, 2009. CBGA is a centralbranch-point intermediate for the biosynthesis of the different classesof cannabinoids. Cyclization of CBGA yields Δ⁹-tetrahydrocannabinolicacid (THCA) or its isomers cannabidiolic acid (CBDA) or cannabichromenicacid (CBCA) (see FIG. 1). The Shoyama group has previously published theidentification and purification of the three enzymes responsible forthese cyclizations (Morimoto S, Komatsu K, Taura F, Shoyama, Y. (1998)Purification and characterization of cannabichromenic acid synthase fromCannabis sativa. Phytochemistry. 49: 1525-1529; Taura F, Morimoto S,Shoyama Y. (1996) Purification and characterization ofcannabidiolic-acid synthase from Cannabis sativa L. Biochemical analysisof a novel enzyme that catalyzes the oxidocyclization of cannabigerolicacid to cannabidiolic acid. J Biol Chem. 271: 17411-17416; and Taura F,Morimoto S, Shoyama Y, Mechoulam R. (1995) First direct evidence for themechanism of 1-tetrahydrocannabinolic acid biosynthesis. J Am Chem Soc.117: 9766-9767). Cloning of THCA and CBDA synthases has also beenpreviously published (Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y,Morimoto S, Shoyama Y. (2005) Tetrahydrocannabinolic acid synthase, theenzyme controlling marijuana psychoactivity, is secreted into thestorage cavity of the glandular trichomes. Plant Cell Physiol. 46:1578-1582.; Taura F, Sirikantaramas S, Shoyama Y, Yoshikai K, Shoyama Y,Morimoto S. (2007) Cannabidiolic-acid synthase, thechemotype-determining enzyme in the fiber-type Cannabis sativa. FEBSLett. 581: 2929-2934. The genes for THCA synthase and CBDA synthase havebeen reported in Japan (Japanese Patent Publication 2000-078979;Japanese Patent Publication 2001-029082).

Cannabinoids are valuable plant-derived natural products. Genes encodingenzymes involved in cannabinoid biosynthesis will be useful in metabolicengineering of cannabis varieties that contain ultra low levels of THCand other cannabinoids via targeted mutagenesis (e.g. TILLING) or othergene knockout techniques. Such genes may also prove useful for creationof specific cannabis varieties for the production of cannabinoid-basedpharmaceuticals, or for reconstituting cannabinoid biosynthesis inheterologous organisms such as bacteria or yeast, or for producingcannabinoids in cell-free systems that utilize recombinant proteins.

Genes encoding enzymes of cannabinoid biosynthesis can also be useful insynthesis of cannabinoid analogs and synthesis of analogs of cannabinoidprecursors. Cannabinoid analogs have been previously synthesized and maybe useful as pharmaceutical products.

There remains a need in the art to identify enzymes, and nucleotidesequences encoding such enzymes, that are involved in the synthesis ofaromatic polyketides.

SUMMARY OF THE INVENTION

A novel gene from cannabis has now been found which encodes a newpolyketide forming enzyme that, acting together with the aforementionedCannabis sativa polyketide synthase/olivetol synthase enzyme(CsPKS/olivetol synthase), catalyzes the formation of olivetolic acid.This newly discovered enzyme is termed Cannabis sativa olivetolic acidsynthase (CsOAS). The CsPKS/olivetol synthase has polyketide synthaseactivity, while CsOAS functions as a polyketide cyclase to formolivetolic acid.

Thus, in a first aspect of the invention, there is provided an isolatedor purified nucleic acid molecule comprising a nucleotide sequencehaving at least 75% sequence identity to SEQ ID NO: 1, or a codondegenerate sequence thereof.

In a second aspect of the invention, there is provided an isolated orpurified nucleic acid molecule comprising a nucleotide sequence havingat least 75% sequence identity to SEQ ID NO: 3, or a codon degeneratesequence thereof.

In a third aspect of the invention, there is provided an isolated orpurified polypeptide comprising an amino acid sequence having at least85% sequence identity to SEQ ID NO: 2, or a conservatively substitutedamino acid sequence thereof.

In a fourth aspect of the invention, there is provided a vector,construct or expression system comprising a nucleic acid molecule of theinvention.

In a fifth aspect of the invention, there is provided a host celltransformed with a nucleic acid molecule of the invention.

In a sixth aspect of the invention, there is provided a process ofsynthesizing a polyketide comprising: reacting an alkanoyl CoA withmalonyl CoA in presence of a type III polyketide synthase enzyme and thepolypeptide of the invention.

In an seventh aspect of the invention, there is provided a process ofaltering levels of cannabinoid compounds in an organism, cell or tissuecomprising using a nucleic acid molecule of the present invention, or apart thereof, to silence in the organism, cell or tissue a gene thatencodes an enzyme that catalyzes synthesis of an aromatic polyketide.

In an eighth aspect of the invention, there is provided a process ofaltering levels of cannabinoid compounds in an organism, cell or tissuecomprising mutating genes in the organism, cell or tissue, and using thenucleic acid molecule of the present invention to select for organisms,cells or tissues containing mutants or variants of a gene that encodesan enzyme that catalyzes synthesis of an aromatic polyketide.

In a ninth aspect of the invention, there is provided a process ofaltering levels of cannabinoid compounds in an organism, cell or tissuecomprising expressing or over-expressing a nucleic acid molecule of theinvention in the organism, cell or tissue in comparison to a similarvariety of organism, cell or tissue grown under similar conditions butwithout the expressing or over-expressing of the nucleic acid molecule.

In a tenth aspect of the invention, there is provided a process ofsynthesizing a naturally-occurring cannabinoid compound or anon-naturally occurring analog of a cannabinoid compound in an organism,cell or tissue comprising expressing the nucleic molecule of theinvention in the organism, cell or tissue in the presence of a type IIIpolyketide synthase enzyme, an alkanoyl CoA and malonyl CoA

In an eleventh aspect of the present invention, there is provided aprocess of synthesizing a polyketide in an in vitro cell-free reaction,said process comprising: reacting acyl carboxylic acids with coenzyme Athrough the action of an acyl CoA synthetase to form alkanoyl CoAs inpresence of a type III polyketide synthase enzyme and the polypeptide ofthe invention.

Polypeptides that are enzymes catalyzing the synthesis of polyketides,and nucleotide sequences encoding such enzymes, have now been identifiedand characterized. As well, synthetic versions of these nucleic acidshave been designed and synthesized. The nucleotide sequences may be usedto create, through breeding, selection or genetic engineering, cannabisplants that overproduce or under-produce cannabinoid compounds, analogsof cannabinoid compounds or mixtures thereof. These nucleotide sequencesmay also be used, alone or in combination with genes encoding othersteps in cannabinoid synthesis pathways, to engineer cannabinoidbiosynthesis in other plants or in microorganisms (e.g. yeast, bacteria,fungi) or other prokaryotic or eukaryotic organisms or in cell-freesystems. In addition, knocking out this gene in cannabis could be usedto block cannabinoid biosynthesis and thereby reduce production ofcannabinoids.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, embodimentsthereof will now be described in detail by way of example, withreference to the accompanying drawings, in which:

FIG. 1 depicts a proposed pathway leading to the main cannabinoid typesin Cannabis sativa. The production of side-products by the polyketidesynthase is shown. Abbreviations: THCA synthase isΔ⁹-tetrahydrocannabinolic acid synthase; CBDA synthase is cannabidiolicacid synthase; CBCA synthase is cannabichromenic acid synthase.

FIGS. 2A-2C depicts liquid chromatography—mass spectrometry (LC-MS)analysis of the enzymatic activity of Cannabis sativa polyketidesynthase/olivetol synthase (CsPKS/olivetol synthase) and Cannabis sativaolivetolic acid synthase (CsOAS). The elution was monitored on a Waters3100 MS in SIR ES⁺mode at 224.95 Da, which detects HTAL and olivetolicacid but not PDAL or olivetol. FIG. 2A depicts an assay ofCsPKS/olivetol synthase with hexanoyl CoA and malonyl CoA in which HTAL(5.2 min) and an unknown compound at 5.9 min were detected. FIG. 2Bdepicts an assay of recombinant CsOAS with hexanoyl CoA and malonyl CoAin which no products were detected. FIG. 2C depicts an assay ofCsPKS/olivetol synthase and CsOAS with hexanoyl CoA and malonyl CoA inwhich, in addition to HTAL (5.2 min) and an unknown compound (5.9 min),a peak corresponding to olivetolic acid was observed at 9.0 minutes.

FIGS. 3A and 3B depict liquid chromatography—photodiode array (PDA)analyses of the enzymatic activity of Cannabis sativa polyketidesynthase/olivetol synthase (CsPKS/olivetol synthase) alone and togetherwith Cannabis sativa olivetolic acid synthase (CsOAS) using hexanoyl CoAas substrate. These assays made use of the recombinant enzyme malonylCoA synthetase (MCS) to produce malonyl CoA. FIG. 3A depicts an assay ofCsPKS/olivetol synthase with hexanoyl CoA in which no olivetolic acidwas detected but HTAL, PDAL and olivetol were present. FIG. 3B depictsan assay of CsPKS/olivetol synthase and CsOAS with hexanoyl CoA in whicholivetolic acid was observed at 9 minutes in addition to HTAL, PDAL andolivetol.

FIGS. 4A-4B depict liquid chromatography—photodiode array (PDA) analysisof the enzymatic activity of Cannabis sativa polyketidesynthase/olivetol synthase (CsPKS/olivetol synthase) alone and togetherwith Cannabis sativa olivetolic acid synthase (CsOAS) using butyryl-CoAas substrate. These assays made use of the recombinant enzyme malonylCoA synthetase (MCS) to produce malonyl CoA. FIG. 4A depicts an assay ofCsOAS with butyryl CoA in which no resorcinolic acid analogs ofolivetolic acid were detected. FIG. 4B depicts an assay ofCsPKS/olivetol synthase and CsOAS with butyryl CoA in which theolivetolic acid analog 2,4-dihydroxy-6-propylbenzoic acid was observedat 18 minutes.

FIGS. 5A and 5B depicts a liquid chromatography—photodiode array (PDA)analysis of the enzymatic activity of Cannabis sativa polyketidesynthase/olivetol synthase CsPKS/olivetol synthase) alone and togetherwith Cannabis sativa olivetolic acid synthase (CsOAS) using octanoyl CoAas substrate. These assays made use of the recombinant enzyme malonylCoA synthetase (MCS) to produce malonyl CoA. FIG. 5A depicts an assay ofCsOAS with octanoyl CoA in which no resorcinolic acid analogs ofolivetolic acid were detected. FIG. 5B depicts an assay ofCsPKS/olivetol synthase and CsOAS with octanoyl CoA in which theolivetolic acid analog 2,4-dihydroxy-6-heptylbenzoic acid was observedat 9 minutes.

FIG. 6 depicts a liquid chromatography—photodiode array (PDA) analysisof the enzymatic activity of Cannabis sativa polyketidesynthase/olivetol synthase together with a codon optimized Cannabissativa olivetolic acid synthase (CsOAS). This assay made use of therecombinant enzyme malonyl CoA synthetase (MCS) to produce malonyl CoA.The assay used Cannabis sativa polyketide synthase/olivetol synthase,MCS and CsOAS with hexanoyl CoA. Olivetolic acid was observed at 9.5minutes.

DESCRIPTION OF PREFERRED EMBODIMENTS

Some embodiments of the present invention relate to an isolated orpurified nucleic acid molecule having SEQ ID No. 1 or having at least75%, at least 76%, least 77%, at least 78%, at least 79%, at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identity to SEQ IDNO: 1.

As is well known to those of skill in the art, it is possible to improvethe expression of a nucleic acid sequence in a host organism byreplacing the nucleic acids coding for a particular amino acid (i.e. acodon) with another codon which is better expressed in the hostorganism. One reason that this effect arises due to the fact thatdifferent organisms show preferences for different codons. Inparticular, bacterial organisms and yeast organisms prefer differentcodons from plants and animals. The process of altering the sequence ofa nucleic acid to achieve better expression based on codon preference iscalled codon optimization. Statistical methods have been generated toanalyze codon usage bias in various organisms and many computeralgorithms have been developed to implement these statistical analysesin the design of codon optimized gene sequences (Lithwick G, Margalit H(2003) Hierarchy of sequence-dependent features associated withprokaryotic translation. Genome Research 13: 2665-73). Othermodifications in codon usage to increase protein expression that are notdependent on codon bias have also been described (Welch et al. (2009)Design parameters to control synthetic gene expression in Escherichiacoli. PLoS ONE 4: e7002).

Some embodiments of the invention relate to codon optimized nucleic acidmolecules based on the sequence of SEQ ID No. 1. In particular, thepresent invention includes an isolated or purified nucleic acid moleculehaving SEQ ID No. 3 or having at least 75%, at least 76%, at least 77%,at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity to SEQ ID NO: 3.

Further included are nucleic acid molecules that hybridize to the abovedisclosed sequences. Hybridization conditions may be stringent in thathybridization will occur if there is at least a 90%, 95% or 97% sequenceidentity with the nucleic acid molecule that encodes the enzyme of thepresent invention. The stringent conditions may include those used forknown Southern hybridizations such as, for example, incubation overnightat 42° C. in a solution having 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/milliliter denatured,sheared salmon sperm DNA, following by washing the hybridization supportin 0.1× SSC at about 65° C. Other known hybridization conditions arewell known and are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001).

As will be appreciated by the skilled practitioner, slight changes innucleic acid sequence do not necessarily alter the amino acid sequenceof the encoded polypeptide. It will be appreciated by persons skilled inthe art that changes in the identities of nucleotides in a specific genesequence that change the amino acid sequence of the encoded polypeptidemay result in reduced or enhanced effectiveness of the genes and that,in some applications (e.g. anti-sense, co suppression, or RNAi), partialsequences often work as effectively as full length versions. The ways inwhich the nucleotide sequence can be varied or shortened are well knownto persons skilled in the art, as are ways of testing the effectivenessof the altered genes. In certain embodiments, effectiveness may easilybe tested by, for example, conventional gas chromatography. All suchvariations of the genes are therefore included as part of the presentdisclosure.

As will be appreciated by one of skill in the art, the length of thenucleic acid molecule described above will depend on the intended use.For example, if the intended use is as a primer or probe, for examplefor PCR amplification or for screening a library, the length of thenucleic acid molecule will be less than the full length sequence, forexample, 15-50 nucleotides. In these embodiments, the primers or probesmay be substantially identical to a highly conserved region of thenucleic acid sequence or may be substantially identical to either the 5′or 3′ end of the DNA sequence. In some cases, these primers or probesmay use universal bases in some positions so as to be ‘substantiallyidentical’ but still provide flexibility in sequence recognition. It isof note that suitable primer and probe hybridization conditions are wellknown in the art.

Some embodiments relate to an isolated or purified polypeptide havingSEQ ID NO. 2 or having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity to the amino acid sequence as setforth in SEQ ID NO: 2.

Some embodiments relate to a vector, construct or expression systemcontaining an isolated or purified polynucleotide having at least 75%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3. As well, there isprovided a method for preparing a vector, construct or expression systemincluding such a sequence, or a part thereof, for introduction of thesequence or partial sequence in a sense or anti-sense orientation, or acomplement thereof, into a cell.

In some embodiments, the isolated and/or purified nucleic acidmolecules, or vectors, constructs or expression systems comprising theseisolated and/or purified nucleic acid molecules, may be used to createtransgenic organisms or cells of organisms that produce polypeptideswhich catalyze the synthesis of aromatic polyketides. Therefore, oneembodiment relates to transgenic organisms, cells or germ tissues of theorganism comprising an isolated and/or purified nucleic acid moleculehaving at least 75% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.

Preferably, the organism is a plant, microorganism or insect. Plants arepreferably of the genus Cannabis, for example Cannabis sativa L.,Cannabis indica Lam. and Cannabis ruderalis Janisch. Especiallypreferred is Cannabis sativa. Microorganisms are preferably bacteria(e.g. Escherichia coli) or yeast (e.g. Saccharomyces cerevisiae). Insectis preferably Spodoptera frugiperda.

Organisms, cells and germ tissues of this embodiment may have alteredlevels of cannabinoid compounds. With reference to FIG. 1, it will beappreciated by one skilled in the art that expression or over-expressionof the nucleic acid molecules of the invention will result in expressionor over-expression of the enzyme that catalyzes the synthesis ofaromatic polyketides (e.g. olivetolic acid) which may result inincreased production of cannabinoid compounds such as cannabigerolicacid (CBGA), Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid(CBDA), cannabichromenic acid (CBCA), Δ⁹-tetrahydrocannabinol,cannabidiol, cannabichromene, etc. Similarly, depending on the substrateused, expression or over-expression of the nucleic acid molecules of theinvention resulting in expression or over-expression of the enzyme thatcatalyzes the synthesis of aromatic polyketides may result in increasedproduction of analogs of cannabinoid compounds, or analogs of precursorsof such compounds.

Silencing of the gene in the organism, cell or tissue will result inunder-expression of the enzyme which may result in accumulation ofprecursors such as malonyl CoA and hexanoyl CoA, and/or reduction ofcannabinoids such as THCA (the precursor of THC) or CBDA (the precursorof cannabidiol (CBD)).

Expression or over-expression of the nucleic acid molecules of theinvention may be done in combination with expression or over-expressionof one or more other nucleic acids that encode one or more enzymes in acannabinoid biosynthetic pathway. Some examples of other nucleic acidsinclude: acyl CoA synthetase synthetase, a type Ill polyketide synthase,a polyketide cyclase, an aromatic prenyltransferase and acannabinoid-forming oxidocylase. Specific examples of these enzymesinclude hexanoyl CoA synthetase, a type Ill polyketide synthase/olivetolsynthase, a geranylpyrophosphate:olivetolate geranyltransferase, aΔ⁹-tetrahydrocannabinolic acid synthase, a cannabidiolic acid synthaseor a cannabichromenic acid synthase.

Expression or over-expression of the enzyme of the present inventioncompared to a control which has normal levels of the enzyme for the samevariety grown under similar or identical conditions will result inincreased levels of cannabinoid compounds, for example, 1-20%, 2-20%,5-20%, 10-20%, 15-20%, 1-15%, 1-10%, 2-15%, 2-10%, 5-15%, or 10-15%(w/w). Cannabinoids already exceed 25% by dry weight in some cannabisvarieties.

Synthesis of aromatic polyketides in the presence of an enzymepolypeptide of the present invention may be accomplished in vivo or invitro. As previously mentioned, such syntheses in vivo may beaccomplished by expressing or over-expressing the nucleic acid moleculeof the invention in an organism, cell or tissue. The organism, cell ortissue may naturally contain alkanoyl CoA and malonyl CoA, or thealkanoyl CoA and malonyl CoA may be provided to the organism, cell ortissue for uptake and subsequent reaction.

Synthesis in vitro can take place in a cell-free system. As part of anin vitro cell-free system, the alkanoyl CoA and malonyl CoA, thepolyketide synthase/olivetolic acid synthase (CsPKS/olivetol synthase)and the polypeptide of the present invention may be mixed together in asuitable reaction vessel to effect the reaction. In vitro, thepolypeptide of the present invention may be used in combination withother enzymes to effect a complete synthesis of a target compound from aprecursor. For example, such other enzymes may be implicated in acannabinoid biosynthetic pathway as described in FIG. 1.

The polypeptides of the present invention may be used, in vivo or invitro, to synthesize analogs of cannabinoid compounds which are notnaturally occurring in the host species. Such analogs can be producedusing alkanoyl CoA compounds other than those used to produce naturalcannabinoid compounds in plants. For example, when the short-chain acylCoA thioesters butyryl CoA (also called n-butyryl CoA to indicate thatit has a straight chain) and octanoyl CoA (also called n-octanoyl CoA toindicate that it has a straight chain) were used as substrates for thepolypeptides of the present invention, resorcinolic acid analogs ofcannabinoid precursors were synthesized. Use of butyryl CoA assubstrates with CsOAS and CsPKS/olivetol synthase polypeptides producedthe resorcinolic acid 2,4-dihydroxy-6-propylbenzoic acid and use ofoctanoyl CoA produced the resorcinolic acid2,4-dihydroxy-6-heptylbenzoic acid.

Terms

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Alkanoyl CoA: An alkanoyl CoA is an aliphatic carbonyl compound having acoenzyme A moiety bonded to the carbon atom of the carbonyl groupthrough a sulfide bridge. Preferred alkanoyl CoA compounds comprise from2 to 6 carbon atoms in the aliphatic carbonyl part of the compound. Morepreferably, the alkanoyl CoA is CoA-S—C(O)—(CH₂)_(n)—CH₃, where n is aninteger from 0 to 4. Some examples of alkanoyl CoA compounds are acetylCoA, butyryl CoA, hexanoyl CoA and octanoyl CoA. Use of acetyl CoAprovides a methyl side chain to the resulting aromatic polyketide; useof butyryl CoA provides a propyl side chain; and use of hexanoyl CoAprovides a pentyl side chain. Hexanoyl CoA is especially preferred. Ithas been shown that cannabinoids with short side-chains exist incannabis (e.g. tetrahydrocannabivarinic acid having a propyl side-chaininstead of the pentyl side-chain of THC acid (Shoyama Y, Hirano H,Nishioka I. (1984) Biosynthesis of propyl cannabinoid acid and itsbiosynthetic relationship with pentyl and methyl cannabinoid acids.Phytochemistry. 23(9): 1909-1912)).

Codon degeneracy: It will be appreciated that this disclosure embracesthe degeneracy of codon usage as would be understood by one of ordinaryskill in the art and as illustrated in Table 1.

TABLE 1 Codon Degeneracies Amino Acid Codons Ala/A GCT, GCC, GCA, GCGArg/R CGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC Asp/D GAT, GAC Cys/CTGT, UGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/HCAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, CTG Lys/KAAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/S TCT,TCC, TCA, TCG, AGT, AGC Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/Y TAT,TAC Val/V GTT, GTC, GTA, GTG START ATG STOP TAG, TGA, TAA

Complementary nucleotide sequence: “Complementary nucleotide sequence”of a sequence is understood as meaning any nucleic acid molecule whosenucleotides are complementary to those of a sequence disclosed herein,and whose orientation is reversed (anti-parallel sequence).

Conservative substitutions: It will be understood by one skilled in theart that conservative substitutions may be made in the amino acidsequence of a polypeptide without disrupting the three-dimensionalstructure or function of the polypeptide. Conservative substitutions areaccomplished by the skilled artisan by substituting amino acids withsimilar hydrophobicity, polarity, and R-chain length for one another.Additionally, by comparing aligned sequences of homologous proteins fromdifferent species, conservative substitutions may be identified bylocating amino acid residues that have been mutated between specieswithout altering the basic functions of the encoded proteins. Table 2provides an exemplary list of conservative substitutions.

TABLE 2 Conservative Substitutions Type of Amino Acid SubstitutableAmino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, ThrSulphydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His AromaticPhe, Tyr, Trp

Degree or percentage of sequence homology: The term “degree orpercentage of sequence homology” refers to degree or percentage ofsequence identity between two sequences after optimal alignment.Percentage of sequence identity (or degree of identity) is determined bycomparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

Homologous isolated and/or purified sequence: “Homologous isolatedand/or purified sequence” is understood to mean an isolated and/orpurified sequence having a percentage identity with the bases of anucleotide sequence, or the amino acids of a polypeptide sequence, of atleast about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.6%, or 99.7%. This percentage is purely statistical, and it ispossible to distribute the differences between the two nucleotide oramino acid sequences at random and over the whole of their length.Sequence identity can be determined, for example, by computer programsdesigned to perform single and multiple sequence alignments.

Increasing, decreasing, modulating, altering or the like: As will beappreciated by one of skill in the art, such terms refer to comparisonto a similar variety or strain grown under similar conditions butwithout the modification resulting in the increase, decrease, modulationor alteration. In some cases, this may be an untransformed control, amock transformed control, or a vector-transformed control.

Isolated: As will be appreciated by one of skill in the art, “isolated”refers to polypeptides or nucleic acids that have been “isolated” fromtheir native environment.

Nucleotide, polynucleotide, or nucleic acid sequence: “Nucleotide,polynucleotide, or nucleic acid sequence” will be understood as meaningboth double-stranded or single-stranded in the monomeric and dimeric(so-called in tandem) forms and the transcription products thereof.

Sequence identity: Two amino acid or nucleotide sequences are said to be“identical” if the sequence of amino acids or nucleotides in the twosequences is the same when aligned for maximum correspondence asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), bythe homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

The definition of sequence identity given above is the definition thatwould be used by one of skill in the art. The definition by itself doesnot need the help of any algorithm, said algorithms being helpful onlyto achieve the optimal alignments of sequences, rather than thecalculation of sequence identity.

From the definition given above, it follows that there is a well definedand only one value for the sequence identity between two comparedsequences which value corresponds to the value obtained for the best oroptimal alignment.

Stringent hybridization: Hybridization under conditions of stringencywith a nucleotide sequence is understood as meaning a hybridizationunder conditions of temperature and ionic strength chosen in such a waythat they allow the maintenance of the hybridization between twofragments of complementary nucleic acid molecules.

Methods

Homologs of the CsOAS genes described herein obtained from otherorganisms, for example plants, may be obtained by screening appropriatelibraries that include the homologs, wherein the screening is performedwith the nucleotide sequence of the specific CsOAS genes of theinvention, or portions or probes thereof, or identified by sequencehomology search using sequence alignment search programs such as BLASTor FASTA.

Nucleic acid isolation and cloning is well established. Similarly, anisolated gene may be inserted into a vector and transformed into a cellby conventional techniques which are known to those of skill in the art.Nucleic acid molecules may be transformed into an organism. As known inthe art, there are a number of ways by which genes, vectors, constructsand expression systems can be introduced into organisms, and acombination of transformation and tissue culture techniques have beensuccessfully integrated into effective strategies for creatingtransgenic organisms. These methods, which can be used in the invention,have been described elsewhere (Potrykus I (1991) Gene transfer toplants: Assessment of published approaches and results. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 42:205-225; Vasil I K (1994) Molecularimprovement of cereals. Plant Mol. Biol. 25: 925-937. Walden R,Wingender R (1995) Gene-transfer and plant regeneration techniques.Trends in Biotechnology 13:324-331; Songstad D D, Somers D A, GriesbachR J (1995) Advances in alternative DNA delivery techniques. Plant CellTissue Organ Cult. 40:1-15), and are well known to persons skilled inthe art.

Suitable vectors are well known to those skilled in the art and aredescribed in general technical references such as Pouwels et al.,Cloning Vectors. A Laboratory Manual, Elsevier, Amsterdam (1986).Particularly suitable vectors include the Ti plasmid vectors. Forexample, one skilled in the art will certainly be aware that, inaddition to Agrobacterium mediated transformation of Arabidopsis byvacuum infiltration (Bechtold N, Ellis J, Pelletier G (1993) In plantaAgrobacterium-mediated gene transfer by infiltration of adultArabidopsis thaliana plants. C R Acad Sci Paris, Sciences de la vie/Lifesciences 316: 1194-1199.) or wound inoculation (Katavic V, Haughn G W,Reed D, Martin M, Kunst L (1994) In planta transformation of Arabidopsisthaliana. Mol. Gen. Genet. 245:363-370.), it is equally possible totransform other plant species, using Agrobacterium Ti-plasmid mediatedtransformation (e.g., hypocotyl (DeBlock M, DeBrouwer D, Tenning P(1989) Transformation of Brassica napus and Brassica oleracea usingAgrobacterium tumefaciens and the expression of the bar and neo genes inthe transgenic plants. Plant Physiol. 91:694-701.) or cotyledonarypetiole (Moloney M M, Walker J M, Sharma K K (1989) High efficiencytransformation of Brassica napus using Agrobacterium vectors. Plant CellRep. 8:238-242.) wound infection, particle bombardment/biolistic methods(Sanford J C, Klein T M, Wolf E D, Allen N (1987) Delivery of substancesinto cells and tissues using a particle bombardment process. J. Part.Sci. Technol. 5:27-37.) or polyethylene glycol-assisted, protoplasttransformation methods (Rhodes C A, Pierce D A, Mettler I J, MascarenhasD, Detmer J J (1988) Genetically transformed maize plants fromprotoplasts. Science 240: 204-207).

As will also be apparent to persons skilled in the art, and as describedelsewhere (Meyer P (1995) Understanding and controlling transgeneexpression. Trends in Biotechnology. 13:332-337; Datla R, Anderson J W,Selvaraj G (1997) Plant promoters for transgene expression.Biotechnology Annual Review 3:269-296.), it is possible to utilizepromoters operatively linked to the nucleic acid molecule to direct anyintended up- or down-regulation of transgene expression usingunregulated (i.e. constitutive) promoters (e.g., those based onCaMV35S), or by using promoters which can target gene expression toparticular cells, tissues (e.g., napin promoter for expression oftransgenes in developing seed cotyledons), organs (e.g., roots), to aparticular developmental stage, or in response to a particular externalstimulus (e.g., heat shock).

Promoters for use in the invention may be inducible, constitutive, ortissue-specific or have various combinations of such characteristics.Useful promoters include, but are not limited to constitutive promoterssuch as carnation etched ring virus promoter (CERV), cauliflower mosaicvirus (CaMV) 35S promoter, or more particularly the double enhancedcauliflower mosaic virus promoter, comprising two CaMV 35S promoters intandem (referred to as a “Double 35S” promoter). It may be desirable touse a tissue-specific or developmentally regulated promoter instead of aconstitutive promoter in certain circumstances. A tissue-specificpromoter allows for over-expression in certain tissues without affectingexpression in other tissues.

The promoter and termination regulatory regions will be functional inthe host cell and may be heterologous (that is, not naturally occurring)or homologous (derived from the host species) to the cell and the gene.

The termination regulatory region may be derived from the 3′ region ofthe gene from which the promoter was obtained or from another gene.Suitable termination regions which may be used are well known in the artand include Agrobacterium tumefaciens nopaline synthase terminator(Tnos), A. tumefaciens mannopine synthase terminator (Tmas) and the CaMV35S terminator (T35S). Particularly preferred termination regions foruse in the present invention include the pea ribulose bisphosphatecarboxylase small subunit termination region (TrbcS) or the Tnostermination region. Gene constructs for use in the invention maysuitably be screened for activity by, for example, transformation into ahost plant via Agrobacterium and screening for altered cannabinoidlevels.

The nucleic acid molecules of the invention, or fragments thereof, maybe used to block cannabinoid biosynthesis in organisms that naturallyproduce cannabinoid compounds. Silencing using a nucleic acid moleculeof the invention may be accomplished in a number of ways generally knownin the art, for example, RNA interference (RNAi) techniques, artificialmicroRNA techniques, virus-induced gene silencing (VIGS) techniques,antisense techniques, sense co-suppression techniques and targetedmutagenesis techniques.

RNAi techniques involve stable transformation using RNA interference(RNAi) plasmid constructs (Helliwell C A, Waterhouse P M (2005)Constructs and methods for hairpin RNA-mediated gene silencing inplants. Methods Enzymology 392:24-35). Such plasmids are composed of afragment of the target gene to be silenced in an inverted repeatstructure. The inverted repeats are separated by a spacer, often anintron. The RNAi construct driven by a suitable promoter, for example,the Cauliflower mosaic virus (CaMV) 35S promoter, is integrated into theplant genome and subsequent transcription of the transgene leads to anRNA molecule that folds back on itself to form a double-stranded hairpinRNA. This double-stranded RNA structure is recognized by the plant andcut into small RNAs (about 21 nucleotides long) called small interferingRNAs (siRNAs). The siRNAs associate with a protein complex (RISC) whichgoes on to direct degradation of the mRNA for the target gene.

Artificial microRNA (amiRNA) techniques exploit the microRNA (miRNA)pathway that functions to silence endogenous genes in plants and othereukaryotes (Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D(2006) Highly specific gene silencing by artificial microRNAs inArabidopsis. Plant Cell 18:1121-33; Alvarez J P, Pekker I, Goldshmidt A,Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAsstimulate simultaneous, efficient, and localized regulation of multipletargets in diverse species. Plant Cell 18:1134-51). In this method, 21nucleotide long fragments of the gene to be silenced are introduced intoa pre-miRNA gene to form a pre-amiRNA construct. The pre-amiRNAconstruct is transferred into the organism genome using transformationmethods which would be apparent to one skilled in the art. Aftertranscription of the pre-amiRNA, processing yields amiRNAs that targetgenes which share nucleotide identity with the 21 nucleotide amiRNAsequence.

In RNAi silencing techniques, two factors can influence the choice oflength of the fragment. The shorter the fragment the less frequentlyeffective silencing will be achieved, but very long hairpins increasethe chance of recombination in bacterial host strains. The effectivenessof silencing also appears to be gene dependent and could reflectaccessibility of target mRNA or the relative abundances of the targetmRNA and the hairpin RNA in cells in which the gene is active. Afragment length of between 100 and 800 bp, preferably between 300 and600 bp, is generally suitable to maximize the efficiency of silencingobtained. The other consideration is the part of the gene to betargeted. 5′ UTR, coding region, and 3′ UTR fragments can be used withequally good results. As the mechanism of silencing depends on sequencehomology, there is potential for cross-silencing of related mRNAsequences. Where this is not desirable, a region with low sequencesimilarity to other sequences, such as a 5′ or 3′ UTR, should be chosen.The rule for avoiding cross-homology silencing appears to be to usesequences that do not have blocks of sequence identity of over 20 basesbetween the construct and the non-target gene sequences. Many of thesesame principles apply to selection of target regions for designingamiRNAs.

Virus-induced gene silencing (VIGS) techniques are a variation of RNAitechniques that exploits the endogenous antiviral defenses of plants.Infection of plants with recombinant VIGS viruses containing fragmentsof host DNA leads to post-transcriptional gene silencing for the targetgene. In one embodiment, a tobacco rattle virus (TRV) based VIGS systemcan be used with the nucleotide sequences of the present invention.

Antisense techniques involve introducing into a plant an antisenseoligonucleotide that will bind to the messenger RNA (mRNA) produced bythe gene of interest. The “antisense” oligonucleotide has a basesequence complementary to the gene's messenger RNA (mRNA), which iscalled the “sense” sequence. Activity of the sense segment of the mRNAis blocked by the anti-sense mRNA segment, thereby effectivelyinactivating gene expression. Application of antisense to gene silencingin plants is described in more detail by Stam M, de Bruin R, vanBlokland R, van der Hoorn RA, Mol J N, Kooter J M (2000) Distinctfeatures of post-transcriptional gene silencing by antisense transgenesin single copy and inverted T-DNA repeat loci. Plant J. 21:27-42.

Sense co-suppression techniques involve introducing a highly expressedsense transgene into a plant resulting in reduced expression of both thetransgene and the endogenous gene (Depicker A, Montagu M V (1997)Post-transcriptional gene silencing in plants. Curr Opin Cell Biol. 9:373-82). The effect depends on sequence identity between transgene andendogenous gene.

Targeted mutagenesis techniques, for example TILLING (Targeting InducedLocal Lesions IN Genomes) and “delete-a-gene” using fast-neutronbombardment, may be used to knockout gene function in an organism(Henikoff S, Till B J, Comai L (2004) TILLING. Traditional mutagenesismeets functional genomics. Plant Physiol 135:630-6; Li X, Lassner M,Zhang Y. (2002) Deleteagene: a fast neutron deletion mutagenesis-basedgene knockout system for plants. Comp Funct Genomics. 3: 158-60).TILLING involves treating germplasm or individual cells with a mutagento cause point mutations that are then discovered in genes of interestusing a sensitive method for single-nucleotide mutation detection.Detection of desired mutations (e.g. mutations resulting in theinactivation of the gene product of interest) may be accomplished, forexample, by PCR methods. For example, oligonucleotide primers derivedfrom the gene of interest may be prepared and PCR may be used to amplifyregions of the gene of interest from organisms in the mutagenizedpopulation. Amplified mutant genes may be annealed to wild-type genes tofind mismatches between the mutant genes and wild-type genes. Detecteddifferences may be traced back to the organism which had the mutant genethereby revealing which mutagenized organism will have the desiredexpression (e.g. silencing of the gene of interest). These organisms maythen be selectively bred to produce a population having the desiredexpression. TILLING can provide an allelic series that includes missenseand knockout mutations, which exhibit reduced expression of the targetedgene. TILLING is touted as a possible approach to gene knockout thatdoes not involve introduction of transgenes, and therefore may be moreacceptable to consumers. Fast-neutron bombardment induces mutations,i.e. deletions, in organism genomes that can also be detected using PCRin a manner similar to TILLING.

It will be understood by one of skill in the art that the processes ofthe invention can also be carried out in a cell-free environment in thepresence of one or more acyl CoA synthase enzymes that form alkanoylCoA.

Embodiments of the invention are susceptible to various modificationsand alternative forms in addition to the specific examples includedherein. Thus, embodiments of the invention are not limited to theparticular forms disclosed.

EXAMPLES Example 1: Isolation and Characterization of CsOAS Gene andEnzyme

An Expressed Sequence Tag (EST) catalog from cannabis glandulartrichomes was analyzed for the highly-expressed proteins of unknownfunction. One unigene showed similarity to a “POP3-like protein” fromArabidopsis (GenBank protein Q9LUV2). The sequence of this protein (SEQID NO: 2) and the corresponding open reading frame (ORF) (SEQ ID NO: 1)of the nucleic acid molecule encoding the protein are given below.

Cannabis sativa CsOAS - 303 bp (SEQ ID NO: 1)ATGGCAGTGAAGCATTTGATTGTATTGAAGTTCAAAGATGAAATCACAGAAGCCCAAAAGGAAGAATTTTTCAAGACGTATGTGAATCTTGTGAATATCATCCCAGCCATGAAAGATGTATACTGGGGTAAAGATGTGACTCAAAAGAATAAGGAAGAAGGGTACACTCACATAGTTGAGGTAACATTTGAGAGTGTGGAGACTATTCAGGACTACATTATTCATCCTGCCCATGTTGGATTTGGAGATGTCTATCGTTCTTTCTGGGAAAAACTTCTCATTTTTGACTACACACCACGA AAGCannabis sativa CsOAS - 101 aa (SEQ ID NO: 2)MAVKHLIVLKFKDEITEAQKEEFFKTYVNLVNIIPAMKDVYWGKDVTQKNKEEGYTHIVEVTFESVETIQDYIIHPAHVGFGDVYRSFWEKLLIFDYTPR K

Example 2: Transformation of E. coli Cells with CsOAS and CsPKS/olivetolSynthase Genes

For expression in E. coli cells, the open reading frames ofCsPKS/olivetol synthase and CsOAS were amplified by PCR, cloned intopHIS8/GW for CsPKS/olivetol synthase or pET100 (Invitrogen) for CsOASand transformed into E. coli BL21 (DE3) (Invitrogen). Cloning wasverified by sequencing.

CsOAS was expressed in 200 mL terrific broth culture whileCsPKS/olivetol synthase grown in a 1 L culture. Both cultures wereincubated at 30° C./150 rpm shaking, induced with 0.5 μM IPTG and grownovernight. The cultures were centrifuged at 16,000 g for 20 min, and thepellets lysed by treatment with lysozyme and sonication. The clearedlysates were mixed with Talon resin (200 μL for CsOAS, 1 mL forCsPKS/olivetol synthase; Clontech), washed with 5 mL of His-tag WashBuffer (50 mM Tris-HCl (pH 7), 150 mM NaCl, 20 mM imidazole, 10 mMβ-mercaptoethanol) and the recombinant proteins eluted using His-tagElution Buffer (20 mM Tris HCl (pH 7), 150 mM NaCl, 100 mM imidazole, 10mM β-mercaptoethanol). The eluate was concentrated using a YM10concentrator and the buffer exchanged to Storage Buffer (20 mM HEPES (pH7.5), 25 mM NaCl, 10% glycerol, 5 mM DTT). The final protein solutionswere quantified by using an RC/DC protein assay kit (Bio-Rad) whichfound protein concentrations of 0.5 mg/mL (CsOAS) and 5.6 mg/mL(CsPKS/olivetol synthase). SDS-PAGE gel analysis confirmed the purity ofboth proteins.

Example 3: Biochemical Activity of CsOAS Enzyme

Activity assays were performed in 50 mM HEPES buffer (pH 7.0) in thepresence of 5 mM DTT, 0.2 mM hexanoyl CoA and 0.2 mM malonyl CoA. 25 μgCsPKS/olivetol synthase was used in assays which were conducted withCsPKS/olivetol synthase, whereas 5 μL of water was used as a substitutewhen CsPKS/olivetol synthase was not required. 10 μL of CsOAS was usedin the following experiments, except in the case where CsPKS/olivetolsynthase was assayed alone and 10 μL of water was used instead. Thetotal volume of reactions was 100 μL. Reaction mixtures were incubatedat 37° C. for 60 minutes with shaking. Products were extracted withethyl acetate, dried by vacuum and resuspended in 30 μL methanol.

The products were analyzed by liquid chromatography-mass spectrometry(LC-MS) on a Waters Alliance system with a Waters Symmetry C18 3.5 μmcolumn (2.1×100 mm). Elution was monitored by Waters PDA 2996 at 280 nMand Waters 3100 mass detector in SIR ES⁺ mode for olivetol (180.91 Da),pentyldiacetic acid lactone (PDAL) (182.96 Da), hexanoyltriacetic acidlactone (HTAL) (224.95 Da) and olivetolic acid (224.95 Da). Dwell wasset for 0.010 sec and cone voltages were set to 25 V for 180.91 Da and224.95 Da, and 40 V for 182.96 Da. MS scan was conducted in ES⁺mode formasses between 150 -650 Da, with a centroid scan of 5000 Da/sec. SolventA consisted of 90% water, 10% acetonitrile with 0.1% formic acid.Solvent B consisted of 99.9% acetonitrile with 0.1% formic acid. 10 μLof sample was injected into the LC-MS and eluted isocratically using 70%solvent A for 5 min at 0.2 mL/min flow rate. A gradient continuedtowards 100% solvent B until 17 min was reached. The gradient returnedto 70% solvent A over 3 min, where it was maintained until 25 min tore-equilibrate. Column temperature was 30° C.±5° C.

The results of the assays of recombinant proteins are shown in FIG. 2.Assayed alone, CsPKS/olivetol synthase catalyzed the formation of twopyrones (pentyldiacetic acid lactone (PDAL) and hexanoyltriacetic acidlactone (HTAL)) and olivetol which confirms the findings of Taura etal., 2009. CsOAS alone did not produce any products when tested withhexanoyl CoA and malonyl CoA. However, when CsPKS/olivetol synthase andCsOAS were assayed together, the reaction mixtures contained olivetolicacid (9.0 min). Therefore CsOAS is an enzyme that functions togetherwith CsPKS/olivetol synthase to form olivetolic acid.

Example 4: Biochemical Activity of CsOAS Enzyme

These assays made use of the recombinant enzyme malonyl CoA synthetase(MCS) to produce malonyl CoA. Activity assays were performed in 20 mMHEPES buffer (pH 7.0) in the presence of 5 mM DTT, 0.2 mM hexanoyl CoA,2.5 mM MgCl₂, 0.5 mM ATP, 0.2 mM coenzyme A, 8 mM sodium malonate, 9 μgCsPKS and 11 μg malonyl CoA synthetase (MCS) with and without 16 μg ofCsOAS. The total volume of the reactions was 100 μL. Reaction mixtureswere incubated at 20° C. for 90 minutes with shaking. Products wereextracted with ethyl acetate, dried by vacuum and resuspended in 60 μLof 70% water/30% acetonitrile. The products were analyzed by liquidchromatography-mass spectrometry (LC-MS) on a Waters Alliance systemwith a Waters Symmetry C18 3.5 μm column (2.1×100 mm) using 70% solventA (90% water, 10% acetonitrile, 0.05% formic acid) and 30% solvent B(acetonitrile +0.05% formic acid) as the elution solvent. The results ofthe assays of recombinant proteins are shown in FIG. 3. These resultsshown in FIG. 3A show that CsPKS forms the two pyrones pentyldiaceticlactone (PDAL) and hexanoyltriacetic lactone (HTAL), and olivetol (OL)but not olivetolic acid. Reactions containing both CsPKS and CsOAS,shown in FIG. 3B, yield olivetolic acid (OA) in addition to the otherproducts.

Example 5: Biochemical Activity of CsOAS Enzyme using Butyryl CoA asSubstrate

These assays made use of the recombinant enzyme malonyl CoA synthetase(MCS) to produce malonyl CoA. Activity assays were performed in 20 mMHEPES buffer (pH 7.0) in the presence of 5 mM DTT, 0.2 mM butyryl CoA,2.5 mM MgCl₂, 0.5 mM ATP, 0.2 mM coenzyme A, 8 mM sodium malonate, 9 μgCsPKS and 11 μg malonyl CoA synthetase (MCS) with and without 16 μg ofCsOAS. The total volume of the reactions was 100 μL. Reaction mixtureswere incubated at 20° C. for 90 minutes with shaking. Products wereextracted with ethyl acetate, dried by vacuum and resuspended in 60 μLof 70% water/30% acetonitrile. The products were analyzed by liquidchromatography-mass spectrometry (LC-MS) on a Waters Alliance systemwith a Waters Symmetry C18 3.5 μm column (2.1×100 mm) using 90% solventA (90% water, 10% acetonitrile, 0.05% formic acid) and 10% solvent B(acetonitrile +0.05% formic acid) as the elution solvent. The results ofthe assays of recombinant proteins are shown in FIG. 4.

The assays with butyryl CoA and CsPKS alone are shown in FIG. 4A, whichshows that the two pyrones, 2 a and 2 b, and the resorcinol, 2 c, areformed, but not the olivetolic acid analog, resorcinolic acid 2 d. Thestructure of each of these compounds is shown below.

The assays with butyryl CoA containing CsPKS and CsOAS are shown in FIG.4B, which shows that each of 2 a, 2 b, 2 c, and the olivetolic acidanalog, 2 d, is formed.

Example 6: Biochemical Activity of CsOAS Enzyme using Octanoyl CoA asSubstrate

These assays made use of the recombinant enzyme malonyl CoA synthetase(MCS) to produce malonyl CoA. Activity assays were performed in 20 mMHEPES buffer (pH 7.0) in the presence of 5 mM DTT, 0.2 mM octanoyl CoA,2.5 mM MgCl₂, 0.5 mM ATP, 0.2 mM coenzyme A, 8 mM sodium malonate, 9 μgCsPKS and 11 μg malonyl CoA synthetase (MCS) with and without 16 μg ofCsOAS. The total volume of the reactions was 100 μL. Reaction mixtureswere incubated at 20° C. for 90 minutes with shaking. Products wereextracted with ethyl acetate, dried by vacuum and resuspended in 60 μLof 70% water/30% acetonitrile. The products were analyzed by liquidchromatography-mass spectrometry (LC-MS) on a Waters Alliance systemwith a Waters Symmetry C18 3.5 μm column (2.1×100 mm) using 60% solventA (90% water, 10% acetonitrile, 0.05% formic acid) and 40% solvent B(acetonitrile +0.05% formic acid) as the elution solvent. The results ofthe assays of recombinant proteins are shown in FIG. 5.

The assays with octanoyl CoA and CsPKS alone are shown in FIG. 5A, whichshows that two pyrones, 3 a and 3 b, and the resorcinol, 3 c, are formedbut not the olivetolic acid analog, resorcinolic acid, 3 d. Thestructure of each of these compounds is shown below.

The assays with octanoyl CoA containing CsPKS and CsOAS are shown inFIG. 5B which shows that 3 a, 3 b and the olivetolic acid analog, 3 d,is formed.

Example 7: Design and Synthesis of Codon-Optimized Nucleic Acid EncodingCsOAS Enzyme

The codon-optimized sequence based on SEQ ID No. 1 (OAS opt) wassynthesized using codons known to provide higher expression in E. coli.

The OAS_opt sequence is:

(SEQ ID NO: 3) ATGGCGGTTAAGCACTTGATCGTCCTGAAGTTCAAAGACGAGATTACTGAGGCCCAAAAAGAAGAGTTTTTCAAAACCTACGTGAATCTGGTGAACATCATTCCGGCGATGAAGGACGTTTACTGGGGTAAAGATGTGACCCAGAAGAACAAAGAAGAGGGCTATACCCATATTGTCGAAGTTACGTTTGAGAGCGTCGAAACCATCCAGGACTATATCATTCATCCGGCACACGTTGGCTTCGGTGATGTGTATCGCAGCTTCTGGGAGAAACTGCTGATCTTTGATTACACGCCGCGT AAG.The DNA sequence of OAS_opt is 79% identical to SEQ ID NO: 1.

Example 8: Transformation of E. coli Cells with Codon-Optimized CsOAS

CsOAS_opt was PCR amplified with Phusion polymerase (Finnzymes) from aplasmid clone using the primers CsOAS_opt forward(5′-ATGGCGGTTAAGCACTTGATC-3′) (SEQ ID NO: 4) and CsOAS_opt reverse(5′-TTACTTACGCGGCGTGTAATC-3′) (SEQ ID NO: 5). PCR products were purifiedand cloned into the pCR8/GW/TOPO entry vector (Invitrogen). Aftertransformation into E. coli TOP10 cells (Invitrogen), individual cloneswere verified by sequencing. The CsOAS_opt was recombined into thepHIS8/GW destination vector using LR recombinase (Invitrogen). The LRreaction products were transformed into TOP10 cells and verified bysequencing.

pHIS8/GW-CsOAS_opt was transformed into E. coli Rosetta 2 cells (Merck).An individual colony was used to inoculate 5 mL liquid LB mediumcontaining 50 μg/mL kanamycin and grown overnight at 37° C. This culturewas used to inoculate 500 mL of overnight autoinduction medium (TB brothcontaining 0.05% glucose, 0.2% α-lactose monohydrate, 50 mM Na₂HPO₄, 50mM KH₂PO₄, 25 mM (NH₄)₂SO₄, 1 mM MgSO₄) containing 50 μg/mL kanamycin.The culture was incubated for 16 hours at 30° C. before adding 0.5 mL500 mM IPTG and allowing cultures to grow another 4 hours at 30° C.

Cultures were centrifuged for 10 minutes at 10 000 g at 4° C. Thesupernatant was discarded; the pellet was collected and frozen at −80°C. The pellet was thawed on ice in the presence of 80 mL of His-taglysis buffer (50 mM Tris-HCl (pH 7.0), 500 mM NaCl, 2.5 mM imidazole,750 μg/mL lysozyme, 10 mM β-mercaptoethanol) for 1 hour. The culture wassonicated on ice and the cell debris pelleted by centrifugation at16,000 g for 20 minutes. The lysate (supernatant) was decanted andtumbled at 4° C. for 30 minutes in the presence of 0.5 mL suspensionvolume of Talon resin (Clontech) followed by centrifugation for 2minutes at 1000 g. After removal of the lysate, the Talon resin waswashed using 5 mL of His-tag wash buffer (50 mM Tris-HCl (pH 7.0), 150mM NaCl, 20 mM imidazole, 10 mM β-mercaptoethanol) followed bycentrifugation (1000 g for 30 seconds). This washing process wasrepeated four times. The Talon resin was transferred to a 5 mLgravity-flow column, rinsed with 5mL His-tag wash buffer, and theprotein was eluted using 5 mL of His-tag elution buffer (20 mM Tris-HCl(pH 7.0), 150 mM NaCl, 200 mM imidazole, 10 mM 3-mercaptoethanol). Theflow-through was concentrated to 1 mL using a 15 mL 5000 MWCO AmiconUltra concentrator (Millipore) which was centrifuged for 30 minutes at2800 g at 4° C. Buffer transfer was accomplished by applying the sampleto a 5 mL Zeba Desalt Spin Column (Thermo Scientific) equilibrated withan appropriate storage buffer (20 mM HEPES (pH 7.0), 25 mM NaCl, 10%glycerol, 5 mM DTT) and centrifuging at 1000 g for 2 minutes at 4° C.The 1.5 mL solution was further concentrated to 300 μL using a 0.5 mL 10000 MWCO Amicon Ultra concentrator (Millipore) that was centrifuged for10 minutes at 10 000 g. The protein sample was quantified using an RC/DCprotein assay kit (Biorad). The CsOAS_opt protein was determined to bepure by SDS-PAGE electrophoresis.

Example 9: Biochemical Activity of Codon-Optimized CsOAS

These assays made use of the recombinant enzyme malonyl CoA synthetase(MCS) to produce malonyl CoA. Activity assays were performed in 20 mMHEPES buffer (pH 7.0) in the presence of 5 mM DTT, 0.2 mM hexanoyl CoA,2.5 mM MgCl₂, 0.5 mM ATP, 0.2 mM coenzyme A, 8 mM sodium malonate, 9 μgCsPKS and 11 μg malonyl CoA synthetase (MCS) with 16 μg of CsOAS_opt.The total volume of reactions was 100 μL. Reaction mixtures wereincubated at 20° C. for 90 minutes with shaking. Products were extractedwith ethyl acetate, dried by vacuum and resuspended in 60 μL of 70%water/30% acetonitrile. The products were analyzed by liquidchromatography-mass spectrometry (LC-MS) on a Waters Alliance systemwith a Waters Symmetry C18 3.5 μm column (2.1×100 mm) using 70% solventA (90% water, 10% acetonitrile, 0.05% formic acid) and 30% solvent B(acetonitrile+0.05% formic acid) as the elution solvent. The results ofthe assays of recombinant proteins are shown in FIG. 6. These resultsshow that reactions containing both CsPKS and codon-optimized CsOASyield olivetolic acid (OA) in addition to PDAL, HTAL and olivetol (OT).

The present invention provides genes which encode a polyketide synthaseenzyme from cannabis. These genes could be used to create, throughbreeding, targeted mutagenesis or genetic engineering, cannabis plantswith enhanced cannabinoid production. In addition, inactivating orsilencing a gene of the invention in cannabis could be used to blockcannabinoid biosynthesis and thereby reduce production of cannabinoidssuch as THCA, the precursor of THC, in cannabis plants (e.g. industrialhemp). The genes of the present invention could be used, in combinationwith genes encoding other enzymes in the cannabinoid pathway, toengineer cannabinoid biosynthesis in other plants or in microorganismsor in cell-free systems, or to produce analogs of cannabinoid compoundsor analogs of cannabinoid precursors

Throughout the present disclosure, reference is made to publications,contents of the entirety of each of which are incorporated by thisreference.

Other advantages that are inherent to the invention are obvious to oneskilled in the art. The embodiments are described herein illustrativelyand are not meant to limit the scope of the invention as claimed.Variations of the foregoing embodiments will be evident to a person ofordinary skill and are intended by the inventors to be encompassed bythe following claims.

1-27. (canceled)
 28. A process of synthesizing a naturally-occurringcannabinoid compound or a non-naturally occurring analog of acannabinoid compound in a microorganism comprising expressing a nucleicmolecule that encodes a polypeptide with at least 95% sequence identityto SEQ ID NO: 2 or that is a conservatively substituted amino acidsequence of the sequence set forth in SEQ ID NO: 2 and having polyketidecyclase activity, in the microorganism in the presence of a type IIIpolyketide synthase enzyme, an alkanoyl CoA and malonyl CoA.
 29. Theprocess of claim 28, wherein the microorganism is a yeast or a bacteria.30. The process of claim 28, wherein the microorganism is Saccharomycescerevisiae yeast or E. coli.
 31. The process of claim 28, wherein thenucleic acid molecule is expressed or over-expressed in combination withexpression or over-expression of one or more other nucleic acids thatencode one or more enzymes in a cannabinoid biosynthetic pathway. 32.The process of claim 31, wherein the one or more enzymes in acannabinoid biosynthetic pathway is one or more of an acyl CoAsynthetase, a polyketide cyclase, an aromatic prenyltransferase or acannabinoid-forming oxidocylase.
 33. The process of claim 32, whereinthe one or more enzymes in a cannabinoid biosynthetic pathway is one ormore of a hexanoyl CoA synthetase, a geranylpyrophosphate:olivetolategeranyltransferase, a Δ9-tetrahydrocannabinolic acid synthase, acannabidiolic acid synthase or a cannabichromenic acid synthase.
 34. Theprocess of claim 28, wherein the cannabinoid compound is one or more ofcannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid,cannabichromenic acid, Δ9-tetrahydrocannabinol, cannabidiol orcannabichromene or an analog thereof comprising a side-chain of 1 to 9carbon atoms in length.
 35. The process of claim 28, wherein thealkanoyl CoA is CoA-S—C(O)—(CH2)n-CH3, where n is an integer from 0 to4.
 36. The process of claim 35, wherein the alkanoyl CoA compriseshexanoyl CoA.
 37. The process of claim 28, wherein the type IIIpolyketide synthase enzyme is polyketide synthase/olivetol synthase. 38.The process of claim 28, wherein the nucleic acid molecule has asequence of SEQ ID NO: 1 or
 3. 39. A process of synthesizing apolyketide in an in vitro cell-free reaction, said process comprising:reacting an acyl carboxylic acid with coenzyme A through the action ofan acyl CoA synthetase to form an alkanoyl CoA in presence of a type IIIpolyketide synthase enzyme and a polypeptide with at least 95% sequenceidentity to SEQ ID NO: 2 or that is a conservatively substituted aminoacid sequence of the sequence set forth in SEQ ID NO: 2 and havingpolyketide cyclase activity.
 40. The process of claim 39, furthercomprises reacting the alkanoyl CoA with a malonyl CoA.
 41. The processof claim 40, wherein the alkanoyl CoA is CoA-S—C(O)—(CH₂)_(n)—CH₃, wheren is an integer from 0 to
 6. 42. The process of claim 41, wherein thealkanoyl CoA comprises hexanoyl CoA, butyryl CoA, or octanoyl CoA. 43.The process of claim 40, wherein the type III polyketide synthase enzymeis polyketide synthase/olivetol synthase.
 44. The process of claim 42,wherein the alkanoyl CoA is hexanoyl CoA.
 45. The process of claim 44,wherein the polyketide is olivetolic acid.
 46. The process of claim 42,wherein the alkanoyl CoA is butyryl CoA
 47. The process of claim 42,wherein the alkanoyl CoA is octanoyl CoA.